Amino Acids at Positions 156 and 332 in the E Protein of the West Nile Virus Subtype Kunjin Virus Classical Strain OR393 Are Involved in Plaque Size, Growth, and Pathogenicity in Mice

The West Nile virus (WNV) subtype Kunjin virus (WNVKUN) is endemic to Australia. Here, we characterized the classical WNVKUN strain, OR393. The original OR393 strain contained two types of viruses: small plaque-forming virus (SP) and large plaque-forming virus (LP). The amino acid residues at positions 156 and 332 in the E protein (E156 and E332) of SP were Ser and Lys (E156S/332K), respectively, whereas those in LP were Phe and Thr (E156F/332T). SP grew slightly faster than LP in vitro. The E protein of SP was N-glycosylated, whereas that of LP was not. Analysis using two recombinant single-mutant LP viruses, rKUNV-LP-EF156S and rKUNV-LP-ET332K, indicated that E156S enlarged plaques formed by LP, but E332K potently reduced them, regardless of the amino acid at E156. rKUNV-LP-EF156S showed significantly higher neuroinvasive ability than LP, SP, and rKUNV-LP-ET332K. Our results indicate that the low-pathogenic classical WNVKUN can easily change its pathogenicity through only a few amino acid substitutions in the E protein. It was also found that Phe at E156 of the rKUNV-LP-ET332K was easily changed to Ser during replication in vitro and in vivo, suggesting that E156S is advantageous for the propagation of WNVKUN in mammalian cells.


Introduction
The West Nile Virus (WNV) is the etiological agent of West Nile fever/West Nile encephalitis.Most (~75%) human WNV infections are asymptomatic, and 1 in 150-250 symptomatic cases develops neuroinvasive disorders [1].Approximately 10% of patients with neuroinvasive diseases die; however, the fatality rate is age-dependent and higher in patients over 70 years of age [1].WNV is a mosquito-borne flavivirus and a member of the Japanese encephalitis virus serocomplex, which includes other clinically important human pathogenic viruses, such as the Japanese encephalitis virus, St. Louis encephalitis virus, Usutu virus, and Murry Valley encephalitis virus [1].The WNV was first isolated from a febrile patient in Uganda in 1937 [2].After the 1950s, several small outbreaks of WNV infection occurred in Africa, the Middle East, parts of Europe, and India, and the virus was considered to induce a mild febrile illness (West Nile fever) [3].However, since the 1990s, the number of severe and fatal neurological cases of WNV infection (West Nile encephalitis) has gradually increased.In 1999, a WNV circulating in the Middle East and Northern Africa was introduced into the New Continent and spread rapidly throughout the region [4][5][6].
WNV is transmitted in enzootic cycles involving Culex mosquito vectors and virus reservoir birds, and humans and domestic animals, such as horses, are considered incidental hosts.Humans are infected with WNV by being bitten, mainly by Culex mosquitoes.No specific drugs or vaccines are available for WNV infection in humans.Although several vaccine candidates against WNV are currently being developed, they have not been approved for human use [7].WNV can be classified into nine lineages (L1-L9) [8].L1 is the most widely distributed lineage of WNV [5].L1 strains have been identified in many regions, including the Americas, Africa, Europe, Russia, India, the Middle East, and Australia [9].L1 strains show highly virulent phenotypes and are involved in serious outbreaks in humans.L1 can be subdivided into three sub-lineages (L1a, L1b, and L1c).The WNV NY99 strain, a representative WNV strain isolated during the first WNV outbreak in the USA, with a highly virulent phenotype in mice, belongs to L1a.L1b is composed of a WNV subtype Kunjin virus strain (WNV KUN ), which is unique to Australia and the only WNV lineage present in Australia [10].WNV KUN has also been isolated from Malaysia [11].WNV KUN causes only mild clinical symptoms in humans and horses, and there have been no reports of death among confirmed cases of infection [12].These findings suggest that WNV KUN may be useful for the development of a live-attenuated vaccine against WNV infection [13][14][15].However, an outbreak of encephalitis caused by WNV KUN occurred in horses in Southeastern Australia in 2011, indicating that a virulent WNV KUN had emerged in the area since the early 2010s [16,17].Moreover, Prow et al. suggested that not only less virulent but also highly virulent strains of WNV KUN have circulated in Australia since the 1980s [18], suggesting that the classical WNV KUN strains are not always suitable for the development of live-attenuated WNV vaccines, and comprehensive virulence analysis is also required for the development of vaccines.
The classic WNV KUN strain OR393 was isolated from Culex mosquitoes in Australia in 1974 [19,20].Several reports have demonstrated that the glycosylation of the potential N-glycosylation site (residues 154-156, Asn-Tyr-Ser) in the WNV E protein is partially involved in its infectivity and pathogenicity, though the modification is not required for WNV pathogenicity in birds [8,9,18,[21][22][23][24][25][26].Most WNV strains are glycosylated at position 154 of E, whereas some classical WNV KUN strains are not.Previous sequence analysis of OR393 revealed that the amino acid at position 156 of the E protein (E 156 ) is Phe (Asn-Tyr-Phe), indicating that the E protein of OR393 is not glycosylated as well as less virulent than classic WNV KUN strains [18,19].In this study, we focused on the OR393 strain and examined its in vitro and in vivo properties to assess its utility in the development of a live-attenuated WNV vaccine.

Viruses
The WNV KUN OR393 strain was isolated from Culex mosquitoes in East Kimberley, Western Australia, in 1974 (GenBank accession No. AF196503) [19].Large plaque-forming virus (LP) and small plaque-forming virus (SP) clones of OR393 were obtained using the limiting-dilution method as described previously [27].Complete nucleotide sequences of the LP-F and SP-B clones were determined.A working virus stock was prepared via amplification in Vero cells.

Plaque Formation Assay for Titration of Infectious Viruses and Analysis of Growth Kinetics
Infectious viral titers for each sample were determined using plaque formation assays.Vero cells (approximately 5 × 10 5 /well) were seeded into 12-well culture plates and inoculated with each virus for 1 h at 37 • C. Next, MEM-based overlay medium containing 1% methylcellulose (FUJIFILM Wako Pure Chemical, Osaka, Japan) and 2% FBS was added to the wells, and the cells were incubated for 5 or 6 days at 36-37 • C, after which they were fixed using a 10% formalin-PBS solution and stained with methylene blue.The diameters (width of the core of the comet-shaped plaques) of 10 plaques were measured, and the mean plaque size (mm ± SD) was calculated.Differences in mean plaque sizes were analyzed using Student's t-test.The ability of WNV KUN strains to grow in vitro was analyzed as previously described [28].Briefly, cells were cultured in six-well culture plates and infected with each WNV KUN strain in 3 mL of MEM supplemented with 2% FBS (2F/MEM) at a multiplicity of infection (MOI) of 0.01-0.05plaque-forming units (PFU)/cell.Small aliquots (200 µL) of the media were collected at one-day intervals, and infectious viral titers were determined using a plaque formation assay in Vero cells, as described above.Infectious virus titers in samples from virus-inoculated mice were statistically compared using Graph-Pad Prism version 7 (GraphPad Software, Boston, MA, USA) and the Mann-Whitney U test.Statistical significance was set at p < 0.05.

Immunoblotting
Culture supernatants and cells were collected 24 and 48 h after virus inoculation, and the cells were lysed in RIPA Buffer (Nacalai Tesque).The supernatant and lysate samples were subjected to SDS-PAGE on a 4-12% gradient polyacrylamide gel (Thermo Fisher Scientific, Waltham, MA, USA).Immunoblotting was performed using an anti-WNV E rabbit polyclonal antibody (GTX132052; GeneTex, Irvine, CA, USA).To examine the glycosylation status of E protein, aliquots of the supernatants and cell lysates were treated with endoglycosidase H (Endo H) and peptide N-glycosidase F (PNGase F) for 90 min at 37 • C according to the manufacturer's instructions (New England Biolabs, Ipswich, MA, USA) before Western blotting.

Establishment of a Reverse-Genetics System for the WNV KUN
A reverse-genetics system for the WNV KUN OR393 large-plaque strain (LP-F; Gen-Bank accession no.LC802099) was established as previously described [29], with some modifications (Supplementary Figure S1).Four viral cDNA fragments (A region: 1-3072, B region: 2832-6013, C region: 5721-8913, and D region: 8595-11020) were synthesized and amplified using a PrimeScript II High Fidelity One-Step RT-PCR kit (Takara Bio, Shiga, Japan).Primers used for amplification are listed in Supplementary Table S1.Each of the four PCR products was inserted into the SmaI site of the plasmid pMW119 (Nippon Gene, Tokyo, Japan) using an In-Fusion HD cloning kit (Takara Bio) and then amplified in E. coli STBL2 (Thermo Fisher Scientific, Waltham, MA, USA).The nucleotide sequences of the plasmid clones A KUNV /pMW, B KUNV /pMW, C KUNV /pMW, and D KUNV /pMW were verified prior to the next amplification step.The four fragments were amplified from the plasmid clones via PCR using the Q5 hot-start PCR master mix (New England Biolabs, Ipswich, MA, USA) and then concatenated to form a full-length amplicon via joint PCR using a 5 ′ -terminal primer with a T7 promoter sequence (T7-KUNV_001f) and a 3-terminal primer (KUNV_11020r).The full-length WNV KUN cDNA amplicon was transcribed using mMESSAGEmMACHINE T7 RNA transcription kit (Thermo Fisher Scientific), and after DNase I treatment and RNA purification, the synthesized RNA was transfected into Vero cells using the TransIT-mRNA Transfection kit (Mirus Bio, Madison, WI, USA), and cells were incubated for 6 days.The culture supernatant fluid was recovered, and a small aliquot was inoculated into Vero cells to amplify the recombinant WNV KUN virus rKUNV.The nucleotide sequence of the recombinant virus was determined using Sanger sequencing, and no unintentional nucleotide mutations were detected.

Production of Point Mutant WNV KUN
To produce the point mutant viruses rKUNV-LP-E F156S and rKUNV-LP-E T332K , the A-region clone A KUNV /pMW was amplified via inverse PCR using primers with point mutations U1433C (E F156S ) and C1961A (E T332K ), respectively (Supplementary Table S1).The PCR products were self-ligated and amplified in E. coli.The resultant clones A KUNV_U1433C / pMW and A KUNV_C1961A /pMW were used to produce recombinant WNV KUN mutants, as described above.The nucleotide sequences of the mutant viruses were determined, and no unintentional mutations were detected.

Mouse Challenge Experiment and Sample Collection
Female ddY mice (Japan SLC, Shizuoka, Japan) were used for challenge tests.For neuroinvasive analysis, groups of mice (3 weeks old, n = 6) were inoculated intraperitoneally (i.p.) with 100 µL (5 × 10 4 PFU and 5 × 10 5 PFU) of the virus solution diluted in 0.9% NaCl solution.The mice were observed, and their body weights were measured daily for 20 days after inoculation to assess survival rates.Survival curves were compared using GraphPad Prism version 7 and log-rank (Mantel-Cox) tests.Statistical significance was set at p < 0.05.To analyze neurovirulence, groups of mice (4 weeks old, n = 6) were inoculated intracerebrally (i.c.) with 30 µL (3 × 10 2 PFU and 3 × 10 3 PFU) of the virus solution, and the mice were observed to determine survival rates, as described above.
For growth analysis, groups of mice (n = 5) were inoculated i.p. with 100 µL (1 × 10 5 PFU) of virus solution.The serum, brain, and spleen were collected from mice at 2 and 5 days post-infection, and the infectious titer and RNA levels of the infectious virus in the samples were measured, as described above and below.Tissue weights were determined, and the tissues were homogenized in 500 µL of 2F/MEM for 30 s at 6000 rpm using Precellys Evolution Touch (Bertin Technologies, Montigny-le-Bretonneux, France).The homogenate was used to measure infectious virus titers and viral genomic copy numbers as described above and below.The nucleotide sequences at positions E 156 and E 332 were determined using Sanger sequencing of several brain samples.

Measurement of Viral Genome Copy Number
Total RNA was extracted from the serum samples using a High Pure Viral RNA Purification Kit (Roche Diagnostics, Indianapolis, IN, USA).To measure the total copy number of the viral genome in the cells and supernatant, we used the real-time RT-PCR (TaqMan) method with the probe WNV_3538p and primers WNVcom.3451f and WNVcom.3590r,as described in Supplemental Table S1.Partial cDNA of the WNV KUN pAKUN clone (AY274505, nt 3301-3800) [30] was synthesized in vitro and inserted into the T7 promoter site downstream of the cloning plasmid pTAC-2 (Eurofins Genomics, Tokyo, Japan).Positive control RNA was synthesized from the plasmid using the mMASSAGE mMACHINE T7 kit, as described above.Genome copy numbers were statistically compared using GraphPad Prism version 7. Statistical significance was set at p < 0.05.

WNV KUN OR393 Contained Small-Sized Plaque and Large-Sized Plaque Viruses
A plaque assay was conducted using Vero cells to determine the infectious titer of the WNV KUN OR393 strain (Figure 1A).The original virus solution contained at least two distinct types of viruses: small plaque-forming virus (SP) and large plaque-forming virus (LP).Single-clone viruses were obtained from the original virus solution using the limiting dilution method to determine the nucleotide sequences of the SP and LP variants.Four SP and three LP clones were obtained (Figure 1B).The complete nucleotide sequences of the two clones from each group (SP-A, SP-B, LP-E, and LP-F) were determined (Table 1).There were six nucleotide variations among the clones, but two (nucleotides 1433 and 1961) of the six sites were different between the SP and LP clones; nucleotides 1433 and 1961 were C and A, respectively, in the SP clones, and U and C, respectively, in the LP clones.The two sites were in the E protein-coding region, and amino acid residues at nucleotides 1433 (amino acid 156 in E, E 156 ) and 1961 (amino acid 332 in E, E 332 ) were Ser (E S156 ) and Lys (E K332 ), respectively, in the SP clones, but Phe (E F156 ) and Thr (E T332 ), respectively, in the LP clones.The other two SP and one LP clones also maintained SP-specific (C1433 and A1961) and LP-specific (U1433 and C1961) sequences at these two positions, respectively (Table 1).These results raise the possibility that these two sites may be associated with the differences in plaque morphology between the SP and LP groups.
clones.The other two SP and one LP clones also maintained SP-specific (C143 and LP-specific (U1433 and C1961) sequences at these two positions, respe 1).These results raise the possibility that these two sites may be associated ferences in plaque morphology between the SP and LP groups.

Growth Ability of Small-and Large-Sized Plaque WNVKUN Clones In Vitro
We selected the SP clone SP-B (GenBank accession No. LC802098) and LP-F for further characterization in vitro.The growth rate of SP-B was slightl that of LP-F in Vero, mosquito C6/36, human neuroblastoma IMR-32, and m blastoma Neuro-2a cells (Figure 2).

Growth Ability of Small-and Large-Sized Plaque WNV KUN Clones In Vitro
We selected the SP clone SP-B (GenBank accession No. LC802098) and the LP clone LP-F for further characterization in vitro.The growth rate of SP-B was slightly higher than that of LP-F in Vero, mosquito C6/36, human neuroblastoma IMR-32, and mouse neuroblastoma Neuro-2a cells (Figure 2).

Glycosylation Status of the E Protein of Small-Sized and Large-Sized Plaque WNVKUN Clones
Asn at position E 154 is an N-linked glycosylation site in the WNV E pro amino acid motif from E 154 to E 156 (Asn-Tyr-Ser) is critical for this modifica Ser in the SP clones and Phe in the LP clones (Table 1).SDS-PAGE and Imm yses showed that the E protein of SP-B migrated slower than that of LP-F, su the difference in the migration rate of the E protein between SP-B and LP-F w glycosylation pattern at position E 154 (Figure 3A).To confirm the effect of gly the E protein on the mobility shift, the cell lysate and supernatant samples with two glycosidases, Endo H and PNGase F (Figure 3B,C).PNGase F rem all types of N-linked (Asn-linked) glycosylation, while Endo H removes on nose and some hybrid types of N-linked carbohydrates.SP-B E protein trea enzymes migrated faster than the untreated SP-B E protein.In contrast, t change in the migration rate of the LP-F E protein after treatment with the e thermore, the mobility of the PNGase F-treated SP-B E protein was similar LP-F E protein.These data indicated that Ser at position E 156 is involved in t Values: means ± standard deviation from three independent inoculations.Significance was analyzed using Student's t-test.p-values are also indicated.

Glycosylation Status of the E Protein of Small-Sized and Large-Sized Plaque WNV KUN Clones
Asn at position E 154 is an N-linked glycosylation site in the WNV E protein, and the amino acid motif from E 154 to E 156 (Asn-Tyr-Ser) is critical for this modification.E 156 was Ser in the SP clones and Phe in the LP clones (Table 1).SDS-PAGE and Immunoblot analyses showed that the E protein of SP-B migrated slower than that of LP-F, suggesting that the difference in the migration rate of the E protein between SP-B and LP-F was due to the glycosylation pattern at position E 154 (Figure 3A).To confirm the effect of glycosylation of the E protein on the mobility shift, the cell lysate and supernatant samples were treated with two glycosidases, Endo H and PNGase F (Figure 3B,C).PNGase F removes almost all types of N-linked (Asn-linked) glycosylation, while Endo H removes only high-mannose and some hybrid types of N-linked carbohydrates.SP-B E protein treated with the enzymes migrated faster than the untreated SP-B E protein.In contrast, there was no change in the migration rate of the LP-F E protein after treatment with the enzymes.Furthermore, the mobility of the PNGase F-treated SP-B E protein was similar to that of the LP-F E protein.These data indicated that Ser at position E 156 is involved in the glycosylation of E in the SP-B clone.

Mutations at E 156 and E 332 of the WNVKUN LP Clone Affected Plaque Formation and G In Vitro
To further investigate the role of the amino acid variations found in SP and vitro and in vivo, a reverse-genetics system for the WNVKUN LP-F clone was esta (Figure S1).The plaques formed by the recombinant LP clone (mean diameter ± S ± 0.124 mm) closely resembled those of the LP-F clone (1.06 ± 0.145 mm) in Ve (Figure 4A).Using this system, two mutant WNVKUN LP clones, rKUNV-LP-E F rKUNV-LP-E T332K , were generated (Figures 4A and S1).The plaques formed by rK LP-E F156S (1.74 ± 0.226 mm) were larger than those formed by SP-B (0.71 ± 0.081 m LP-F (Figure 4A).rKUNV-LP-E T332K formed plaques whose size (0.76 ± 0.087 mm) w ilar to that of SP-B.The plaque size of the E 156S/332T virus (rKUNV-LP-E F156S ) was larg that of the E 156F/332T virus (LP virus), but the size of the E 156S/332K virus (SP-B) was equ to that of the E 156F/332K virus (rKUNV-LP-E T332K ).The plaques formed by the E 332K (SP-B and rKUNV-LP-E T332K ) were smaller than those formed by the E 332T viru clones and rKUNV-LP-E F156S ).These results indicate that the amino acid residue was dominant to that of E 156 in regulating the plaque size formed by LP-F, and, th the plaque size is mainly driven by E 332 in SP and LP variants (Figure 4B).The grow of rKUNV-LP-E F156S and rKUNV-LP-E T332K resembled SP-B in Vero, C6/36, and Ne cells (Figure 4C).SP-B and rKUNV-LP-E T332K grew faster than rKUNV-LP and rKUN E F156S in IMR-32 cells.

Mutations at E 156 and E 332 of the WNV KUN LP Clone Affected Plaque Formation and Growth In Vitro
To further investigate the role of the amino acid variations found in SP and LP in vitro and in vivo, a reverse-genetics system for the WNV KUN LP-F clone was established (Figure S1).The plaques formed by the recombinant LP clone (mean diameter ± SD: 1.06 ± 0.124 mm) closely resembled those of the LP-F clone (1.06 ± 0.145 mm) in Vero cells (Figure 4A).Using this system, two mutant WNV KUN LP clones, rKUNV-LP-E F156S and rKUNV-LP-E T332K , were generated (Figure 4A and Figure S1).The plaques formed by rKUNV-LP-E F156S (1.74 ± 0.226 mm) were larger than those formed by SP-B (0.71 ± 0.081 mm) and LP-F (Figure 4A).rKUNV-LP-E T332K formed plaques whose size (0.76 ± 0.087 mm) was similar to that of SP-B.The plaque size of the E 156S/332T virus (rKUNV-LP-E F156S ) was larger than that of the E 156F/332T virus (LP virus), but the size of the E 156S/332K virus (SP-B) was equivalent to that of the E 156F/332K virus (rKUNV-LP-E T332K ).The plaques formed by the E 332K viruses (SP-B and rKUNV-LP-E T332K ) were smaller than those formed by the E 332T viruses (LP clones and rKUNV-LP-E F156S ).These results indicate that the amino acid residue of E 332 was dominant to that of E 156 in regulating the plaque size formed by LP-F, and, therefore, the plaque size is mainly driven by E 332 in SP and LP variants (Figure 4B).The growth rate of rKUNV-LP-E F156S and rKUNV-LP-E T332K resembled SP-B in Vero, C6/36, and Neuro-2A cells (Figure 4C).SP-B and rKUNV-LP-E T332K grew faster than rKUNV-LP and rKUNV-LP-E F156S in IMR-32 cells.S2.

Virulence of the WNV KUN SP and LP Clones and Recombinant WNV KUN Mutants in Mice
We examined the neurovirulence and neuroinvasiveness of the SP, LP, and recombinant mutants in mice.Mice were infected i.c. with SP-B, rKUNV-LP, rKUNV-LP-E F156S , or rKUNV-LP-E T332K .All mice inoculated with 3 × 10 2 PFU of the viruses survived (Figure 5A).In the 3 × 10 3 PFU-inoculated groups, all mice inoculated with rKUNV-LP died at 5 days post-infection, whereas all mice inoculated with SP-B, rKUNV-LP-E F156S , or rKUNV-LP-E T332K died at 6 days post-infection (Figure 5B).
Mice were also infected i.p. with the four viruses.In the group infected with 5 PFU, one (16.7%),three (50%), and four (66.7%) out of six mice inoculated with rKU LP, rKUNV-LP-E T332K , and SP-B, respectively, died within the observation period, wh all rKUNV-LP-E F156S -infected mice died by 10 days post-infection (Figure 5C).In the g infected with 5 × 10 5 PFU, at least four (66.7%) of the six mice inoculated with rKUNV rKUNV-LP-E T332K , or SP-B survived throughout the observation period, but all mice ulated with rKUNV-LP-E F156S died within 9 days post-infection (Figure 5D).Mice were also infected i.p. with the four viruses.In the group infected with 5 × 10 4 PFU, one (16.7%),three (50%), and four (66.7%) out of six mice inoculated with rKUNV-LP, rKUNV-LP-E T332K , and SP-B, respectively, died within the observation period, whereas all rKUNV-LP-E F156S -infected mice died by 10 days post-infection (Figure 5C).In the group infected with 5 × 10 5 PFU, at least four (66.7%) of the six mice inoculated with rKUNV-LP, rKUNV-LP-E T332K , or SP-B survived throughout the observation period, but all mice inoculated with rKUNV-LP-E F156S died within 9 days post-infection (Figure 5D).

Growth of the WNV KUN SP and LP Clones and Recombinant WNV KUN Mutants in Mice
Infectious viruses and viral RNA levels were investigated in mice inoculated i.p. with the recombinant viruses (Figure 6).Two days after inoculation, infectious viruses were detected in most serum and spleen samples, although no infectious viruses were detected in the brains of any of the four groups.High levels of viremia were observed in the sera of mice inoculated with rKUNV-LP-E F156S , rKUNV-LP-E T332K , and SP-B strains (Figure 6A).The levels of viral RNA in the serum samples were also significantly higher in rKUNV-LP-E F156S -, rKUNV-LP-E T332K -, and SP-B-inoculated mice than in rKUNV-LPinoculated animals (Figure S2).In spleen samples, no clear differences in infectious titers were observed among the strains (Figure 6C).Five days after inoculation, the number of infectious viruses decreased and was not observed in half of the serum samples from any of the four groups (Figure 6A).In the brain samples, significantly higher levels of the infectious virus were detected in the rKUNV-LP-E F156S -inoculated group (Figure 6B).In contrast, the level of infectious viruses in SP-B-infected mice was higher than that in rKUNV-LP-E F156Sinfected mice (Figure 6C).Samples from rKUNV-LP-infected mice showed lower levels of viremia and infectious viruses than those from other virus-infected groups.

Growth of the WNVKUN SP and LP Clones and Recombinant WNVKUN Mutants in Mi
Infectious viruses and viral RNA levels were investigated in mice inoculated i.p the recombinant viruses (Figure 6).Two days after inoculation, infectious viruses detected in most serum and spleen samples, although no infectious viruses were de in the brains of any of the four groups.High levels of viremia were observed in th of mice inoculated with rKUNV-LP-E F156S , rKUNV-LP-E T332K , and SP-B strains (Figu The levels of viral RNA in the serum samples were also significantly higher in rK LP-E F156S -, rKUNV-LP-E T332K -, and SP-B-inoculated mice than in rKUNV-LP-inoculat imals (Figure S2).In spleen samples, no clear differences in infectious titers were obs among the strains (Figure 6C).Five days after inoculation, the number of infectio ruses decreased and was not observed in half of the serum samples from any of th groups (Figure 6A).In the brain samples, significantly higher levels of the infectious were detected in the rKUNV-LP-E F156S -inoculated group (Figure 6B).In contrast, th of infectious viruses in SP-B-infected mice was higher than that in rKUNV-LP-E F fected mice (Figure 6C).Samples from rKUNV-LP-infected mice showed lower lev viremia and infectious viruses than those from other virus-infected groups.or rKUNV-LP-E T332K (n = 5) were euthanized at 2 or 5 days after inoculation, and serum (A (B), and spleen (C) samples were collected.Sera and tissue homogenates were used to quan infectious virus titer (PFU/mL or g).Dotted line: detection limit.Geometric mean titers and g ric standard deviations are indicated by horizontal bars.Significance was analyzed using the Whitney U test (* p < 0.05, **p < 0.01).

Genomic Stability of the E 156 and E 332 Mutations in the Recombinant WNVKUN Mutan
We confirmed the N-glycosylation of recombinant WNVKUN E proteins in Ver via immunoblot analysis (Figure 7A).The E protein of rKUNV-LP-E F156S , similar to migrated more slowly than rKUNV-LP, and the mobility of the PNGase F-treated rK LP-E F156S E protein was similar to that of the rKUNV-LP E protein, indicating th rKUNV-LP-E F156S E protein was glycosylated in Vero cells.However, two di or rKUNV-LP-E T332K (n = 5) were euthanized at 2 or 5 days after inoculation, and serum (A), brain (B), and spleen (C) samples were collected.Sera and tissue homogenates were used to quantify the infectious virus titer (PFU/mL or g).Dotted line: detection limit.Geometric mean titers and geometric standard deviations are indicated by horizontal bars.Significance was analyzed using the Mann-Whitney U test (* p < 0.05, ** p < 0.01).

Genomic Stability of the E 156 and E 332 Mutations in the Recombinant WNV KUN Mutants
We confirmed the N-glycosylation of recombinant WNV KUN E proteins in Vero cells via immunoblot analysis (Figure 7A).The E protein of rKUNV-LP-E F156S , similar to SP-B, migrated more slowly than rKUNV-LP, and the mobility of the PNGase F-treated rKUNV-LP-E F156S E protein was similar to that of the rKUNV-LP E protein, indicating that the rKUNV-LP-E F156S E protein was glycosylated in Vero cells.However, two different migration signals, rKUNV-LP-like and SP-B-like (slow) patterns, were observed in rKUNV-LP-E T332K -infected cell samples, and the SP-B-like pattern disappeared after treatment with PNGase F. Nucleotide sequences at sites E 156 and E 332 were determined in rKUNV-LP-E T332K passaged once, twice, and three times in Vero cells (Figure 7B).No mutation at E 332 was observed in the three viruses, whereas the amino acid residue at E 156 was partially changed from Phe to Ser (from U to C at nucleotide position 1433) in the twice-passaged virus and completely changed in the three-times-passaged virus.We also examined the nucleotide sequences of sites E 156 and E 332 in day 5 mouse brain samples used for the growth analysis shown in Figure 6 (Table S3).The amino acid residue at E 156 of the virus detected in the rKUNV-LP-E T332K -infected mouse brains was Ser (C at nucleotide position 1433) in all three samples examined.Partial amino acid changes from Phe to Ser were also observed in the brain samples of mice infected with rKUNV-LP.
rKUNV-LP-E passaged once, twice, and three times in Vero cells (Figure 7B).No mutation at E 332 was observed in the three viruses, whereas the amino acid residue at E 156 was partially changed from Phe to Ser (from U to C at nucleotide position 1433) in the twicepassaged virus and completely changed in the three-times-passaged virus.We also examined the nucleotide sequences of sites E 156 and E 332 in day 5 mouse brain samples used for the growth analysis shown in Figure 6 (Table S3).The amino acid residue at E 156 of the virus detected in the rKUNV-LP-E T332K -infected mouse brains was Ser (C at nucleotide position 1433) in all three samples examined.Partial amino acid changes from Phe to Ser were also observed in the brain samples of mice infected with rKUNV-LP.

Discussion
In this study, we investigated the characteristics of the WNVKUN OR393 strain, which was isolated from Culex mosquitoes in Australia in the 1970s, to evaluate the possibility of using this virus as a candidate backbone to develop a live-attenuated WNV vaccine.However, the original stock of the virus was mixed with two substrains (LP and SP) with different plaque-formation abilities in Vero cells.
We obtained several clones of LP and SP, and their nucleotide sequences indicated that the two amino acid residues at E 156 and E 332 are involved in the plaque phenotype.Adams et al. showed that the amino acid residue of E 156 of OR393 was phenylalanine [19], suggesting that the mutation at E 156 may have occurred during the process of virus passage, although the exact passage history is unknown.As mentioned later, we proved in this study that the mutation at E 156 occurs easily by passaging rKUNV-LP-E T332K in Vero

Discussion
In this study, we investigated the characteristics of the WNV KUN OR393 strain, which was isolated from Culex mosquitoes in Australia in the 1970s, to evaluate the possibility of using this virus as a candidate backbone to develop a live-attenuated WNV vaccine.However, the original stock of the virus was mixed with two substrains (LP and SP) with different plaque-formation abilities in Vero cells.
We obtained several clones of LP and SP, and their nucleotide sequences indicated that the two amino acid residues at E 156 and E 332 are involved in the plaque phenotype.Adams et al. showed that the amino acid residue of E 156 of OR393 was phenylalanine [19], suggesting that the mutation at E 156 may have occurred during the process of virus passage, although the exact passage history is unknown.As mentioned later, we proved in this study that the mutation at E 156 occurs easily by passaging rKUNV-LP-E T332K in Vero cells (Figure 7).Analysis using recombinant WNV KUN mutants clearly showed that the E 156S virus formed larger plaques than the E 156F virus when the residue E 332 was Thr.The WNV E protein is composed of three structural domains: I, II, and III (EDI, EDII, and EDIII) [31].E 156 is located on the N-glycosylation motif (Asn-Tyr-Ser) in the EDI, and this residue influences the N-glycosylation of the Asn residue at E 154 , suggesting that glycosylation is associated with the plaque morphology of WNV KUN .We confirmed that the E protein of SP-B is N-glycosylated, whereas that of LP-F is not.However, E 332K potently decreased the plaque size, regardless of the residue at position E 156 .These results indicate that the residue of E 332 is a dominant determinant of plaque size.The LP strains (LP-F and rKUNV-LP) grew slower than the other strains, indicating that the combination of E 156F and E 332T decreased virus growth in cultured cells.The threonine residue at E 332 is conserved among WNV KUN , and the Lys residue at this position is unique to SP-B (Figure S3).Although the reason why E 332K emerged during passaging in the mouse brain and Vero cells remains unknown, our data imply that E 332K may be advantageous for growth in Vero cells or the mouse brain when the residue in E 156 is phenylalanine rather than serine.Our plaque and growth analyses in Vero cells also demonstrated that the growth rate is not necessarily correlated with plaque size in Vero cells among OR393 substrains.
The survival curves of the four WNV KUN strains in the i.c.inoculation experiments were similar, and all infected mice died 6 days post-infection, suggesting that these viruses have equivalent neurovirulence in mice.However, all rKUNV-LP-inoculated mice died 5 days post-infection, whereas mice inoculated with the other strains died 6 days postinfection in the 3 × 10 3 PFU/mouse group.Moreover, in the 1.5 × 10 4 PFU/mouse inoculation, all rKUNV-LP-inoculated mice died at 5 days post-infection; however, most of the mice inoculated with the other strains died at 6 days post-infection (Figure S4).These results were unexpected because previous reports have revealed that E 156S in WNV is a virulent type, but E 156F is not.An analysis using chimeric viruses between the virulent WNV and non-pathogenic WNV KUN also suggested that not only E 156 but also other regions of the E protein are important for the pathogenicity of WNV [25].E 332 is located in EDIII, which forms an immunoglobulin-like domain that is thought to play a crucial role in receptor binding and viral attachment to the cell surface (Figure S5) [32].Mutations in EDIII result in altered virulence, suggesting that this domain is involved in viral pathogenesis [33,34].
In contrast, the results of i.p. inoculation indicated that rKUNV-LP-E F156S exhibited a significantly higher neuroinvasive ability than the other three strains.Furthermore, the infectious virus levels in rKUNV-LP-E F156S -infected mouse brains were higher than those in mice infected with other viruses.These results demonstrated that the E 156S/332T -type virus has the potential to increase the neuroinvasiveness of WNV KUN .Previous studies have indicated the importance of N-glycosylation of E protein in the neurovirulence and neuroinvasiveness of WNV in mammalian hosts [8,9,[21][22][23]25]. Glycosylation influences virus binding to cell surface attachment factors and the infectivity of WNV [24].WNV strains containing N-glycosylation at E 154 use DC-SIGN, a C-type lectin present on the surface of dendritic cells, as an attachment factor to enhance infection compared with nonglycosylated strains [35].DC-SIGNR also promotes WNV infection more efficiently than DC-SIGN in mammalian cells, and this effect is dependent on N-glycosylation [36].These previous findings may help understand the basis of the increased growth and pathogenicity of the E 156S/332T -type WNV KUN strains.In contrast, E 156S/332K -type SP-B resulted in lower viremia levels and neuroinvasiveness than E 156S/332T -type rKUNV-LP-E F156S in mice.The E 156F/332T -type rKUNV-LP showed the lowest neuroinvasiveness and infectious virus levels in the serum, brain, and spleen of mice, implying that, in contrast to the results of the neurovirulence analysis, the combination of E 156F and E 332T may be negatively associated with neuroinvasiveness in mice.Thus, our data suggest that the amino acid residues at E 156 and E 332 of WNV KUN play different roles in neurovirulence and neuroinvasiveness. Furher comprehensive analyses are required to understand the mechanisms of action of these residues.Generally, pathogenic WNV produces large plaques, which are indicative of rapid cell proliferation.However, our data demonstrated that the plaque size formed by infection with WNV cannot be used as an indicator of its virulence [37].Our results also showed that low-pathogenic classical strains of WNV KUN could be easily transformed into highly pathogenic viruses by only a few amino acid substitutions in the E protein.The outbreak of WNV in horses in Australia in the 2010s may have been caused by several mutations in the WNV KUN genome that made the virus more virulent [18].It is important to continue to analyze the genome of the virus and pay attention to the genome sequences of WNV KUN .
Our data in Figure 7 and Table S3 show that the Phe residue at E 156 in rKUNV-LP-E T332K was rapidly replaced with Ser in Vero cells and in mice.This implies that rKUNV-LP-E T332K is changed to the SP (E 156S/332K ) virus by passaging in these cells.The plaque

Figure 3 .
Figure 3. Immunoblot analysis of culture supernatant and cell lysate of SP-B-and LP-F-infec cells.(A) WNVKUN E protein in the samples was detected with an anti-WNV E antibody GTX (B,C) Cell lysates (B) and culture supernatants (C) were treated with endoglycosidase H (E and peptide N-glycosidase F (PNGase F) before loading onto an SDS-PAGE gel.WNVKUN E in the samples was detected with the anti-WNV E antibody.Mock indicates mock-inoculat ples.(-) indicates non-glycosidase reaction control.Markers indicate molecular weight mar

Figure 3 .
Figure 3. Immunoblot analysis of culture supernatant and cell lysate of SP-B-and LP-F-infected Vero cells.(A) WNV KUN E protein in the samples was detected with an anti-WNV E antibody GTX132052.(B,C) Cell lysates (B) and culture supernatants (C) were treated with endoglycosidase H (Endo H) and peptide N-glycosidase F (PNGase F) before loading onto an SDS-PAGE gel.WNV KUN E protein in the samples was detected with the anti-WNV E antibody.Mock indicates mock-inoculated samples.(-) indicates non-glycosidase reaction control.Markers indicate molecular weight markers.

Figure 7 .
Figure 7. (A) Immunoblot analysis of cell lysates from recombinant WNVKUN-infected Vero cells.Cells were collected 48 h after virus inoculation and lysed.The cell lysate was treated with PNGase F before loading onto SDS-PAGE gel.WNVKUN E protein in the samples was detected using an anti-WNV E antibody.SP-B-infected cell samples were used as N-glycosylation-positive controls.Mock indicates mock-infected Vero cell lysates.(−): no PNGase F-treated lysate.(B) Amino acid residues at E 156 and E 332 in rKUNV-LP-E T332K viruses passaged repeatedly in Vero cells.

Figure 7 .
Figure 7. (A) Immunoblot analysis of cell lysates from recombinant WNV KUN -infected Vero cells.Cells were collected 48 h after virus inoculation and lysed.The cell lysate was treated with PNGase F before loading onto SDS-PAGE gel.WNV KUN E protein in the samples was detected using an anti-WNV E antibody.SP-B-infected cell samples were used as N-glycosylation-positive controls.Mock indicates mock-infected Vero cell lysates.(−): no PNGase F-treated lysate.(B) Amino acid residues at E 156 and E 332 in rKUNV-LP-E T332K viruses passaged repeatedly in Vero cells.

Table 1 .
Nucleotide and amino acid variations in small-and large-plaque clones iso WNV KUN OR393 strain.

Table 1 .
Nucleotide and amino acid variations in small-and large-plaque clones isolated from the WNV KUN OR393 strain.